Therapy of cancer remains one of the major challenges of medicine today. Only a combined therapeutic approach will allow this problem to be mastered. This will involve surgery, classical chemotoxic chemotherapy, molecular targeted drugs and immunotherapy.
The major problem in the use of chemotoxic drugs is their low selectivity for cancer cells resulting in dose limiting and life threatening toxic side effects. The most common acute toxicity is myelotoxicity resulting in a severe leukopenia and thrombocytopenia. Some of the commonly used drugs have also a more specific toxicity. Doxorubicin (Dox), an anthracycline drug, is an 17 of such a chemotoxic drug that induces besides severe myelotoxicity a severe cardiotoxicity. These toxicities restrict its use above a cumulative dose of 500 mg/m2.
Approaches used to increase tumor specificity of a drug are conjugation with (i) a tumor-recognizing molecule (e.g. receptor ligand; see, e.g., Safavy et al. 1999—J Med Chem 42,4919-4924) or with (ii) a peptide that is cleaved preferentially in the immediate vicinity of tumor cells by proteases preferentially secreted or produced by tumor cells.
Tumor specific oligopeptidic prodrugs, such as prodrugs of doxorubicin, have been developed. In contrast to previous studies, these peptidic prodrugs were designed to be impermeable to cell membranes, to remain stable in the blood while being cleaved into the active drug by peptidases released in the extracellular space of solid tumors. These activating peptidases are not necessarily tumor specific but can increase the drug selectivity to the extent that these peptides are oversecreted in the extracellular space of solid tumors and play an important role in cancer cell invasion and metastasis. The originality of this approach is that it didn't target a single well known enzyme but all enzymatic activity that was found excreted by human tumoral cells maintained in culture. N-succinyl-beta-alanyl-L-leucyl-L-alanyl-L-leucyl-doxorubicin (Suc-βALAL-dox) was selected as such a candidate prodrug (Fernandez et al. 2001, J Med Chem 44:3750-3). Compared with unconjugated doxorubicin this prodrug is, in mice, about 5 times, and in dogs, 3 times less toxic. Chronic treatment with Suc-βALAL-dox proved to be significantly less cardiotoxic than with Dox at doses up to 8-fold higher in rats. The improved activity of Suc-βALAL-dox over Dox was observed in several tumor xenograft models (Dubois et al. 2002, Cancer Res 62:2327-31; Ravel et al. 2008, Clin Cancer Res 14:1258-65). Two enzymes, CD10 (neprilysin or calla antigen) and thimet oligopeptidase (TOP) have been identified later in tumor cell conditioned medium and in tumor cells as activators of Suc-βALAL-dox (Pan et al. 2003, Cancer Res 63:5526-31; Dubois et al. 2006, Eur J Cancer 42:3049-56) but other non-identified proteases may also be involved in the activation process.
A phase I clinical study with Suc-βALAL-dox was initiated by the biopharmaceutical company DIATOS SA. A myelotoxicity occurred at three times higher doses compared with Dox. No drug-related, severe cardiac adverse events were reported, even at very high cumulative doses (2750 mg/m2). A clinical benefit was observed for 59% of evaluable patients (Delord et al., unpublished).
The main limitation of Suc-βALAL-dox is that leukopenia remains as an important toxicity and that experimentally higher antitumoral activity could only be observed at the cost of a still important myelotoxicity. Such myelotoxicity is expected to occur as the result from the sensitivity of the peptidic moiety of Suc-βALAL-dox to the hydrolysis by enzymes present in normal tissues.
WO 02/100353 specifically discloses chemotherapeutic prodrugs designed with a 3- to 6-amino acid oligopeptide cleavable by CD10. WO 02/00263 discloses prodrugs with a 3-amino acid oligopeptide cleavable by TOP and at least 1 prodrug with an amino acid oligopeptide (Leu-Ala-Gly) not cleavable by CD10. WO 00/33888 and WO 01/95945 disclose prodrugs with a 4- to 20-amino acid oligopeptide comprising a non-genetically encoded amino acid at a fixed position, with said oligopeptide being cleavable by TOP. In WO 01/95945, at least 1 prodrug, with a βAla-Leu-Tyr-Leu oligopeptide, was reported to be resistant to CD10 proteolytic action. WO 01/95943 discloses prodrugs with a 3- to 4-amino acid oligopeptide comprising a fixed isoleucine, said oligopeptide preferably being resistant to TOP; no information on CD10-susceptibility or -resistance is given. A more general concept of a prodrug consisting of a drug linked to an oligopeptide (of at least 2 amino acids) itself linked to a terminal group is disclosed in WO 96/05863 and was later extended in WO 01/91798.
Other polymeric drug-conjugates of which the non-drug moiety is at least comprising a water-soluble polymer and a peptide (comprising 4 to 5 natural or non-natural amino acids) selectively cleavable by action of matrix metalloproteinases (MMPs) are disclosed in WO 02/07770. WO 03/094972 focuses on anti-tumor prodrugs that are activatable by the human fibroblast activation protein (FAPα); the prodrug comprises an oligopeptide of 4 to 9 amino acids with a cyclic amino acid at a fixed position. WO 99/28345 discloses prodrugs that are proteolytically cleavable by prostate-specific antigen in the oligopeptide of less than 10 amino acids present in the prodrug.
WO 97/34927 revealed the FAPα-scissable prodrugs Ala-Pro-7-amino-4-trifluoromethylcoumarin and Lys-Pro-7-amino-4-trifluoromethylcoumarin. WO 00/71571 focuses on FAPα-scissable prodrugs, with some further experimental investigations on proteolytic sensitivity to CD26 (dipeptidylpeptidase IV), the latter being considered as undesirable due to the relative abundance of CD26 also in non-malignant cells.
Other prodrugs activatable by FAPα include promellitin toxin (LeBeau et al. 2009, Mol Cancer Ther 8, 1378-1386), doxorubicin (Huang et al. 2011, J Drug Target 19, 487-496), thapsigargin (Brennen et al. 2012, J Natl Cancer Inst 104, 1320-1334), and a prodrugs comprising an oligopeptide of 4 to 9 amino acids with a cyclic amino acid at a fixed position (WO 03/094972). WO 01/68145 discloses MMP-cleavable but neprilysin (CD10)-resistant doxorubicin prodrugs (see Example 1001 therein) comprising a 3- to 8-amino acid oligopeptide. Metalloproteinase- and plasmin-sensitive doxorubicin prodrugs have been developed, as well as CNGRC-peptide conjugates with doxorubicin (Hu et al. 2010, Bioorg Med Chem Lett 20, 853-856; Chakravarty et al. 1983, J Med Chem 26, 638-644; Devy et al. 2004, FASEB J 18, 565-567; Vanhensbergen et al. 2002, Biochem Pharmacol 63, 897-908).
WO97/12624, WO97/14416, WO98/10651, WO98/18493 and WO99/02175 disclose peptide-comprising prodrugs wherein the peptide is cleavable by the prostate-specific antigen (PSA).
Common to all above prodrugs is the presence of a protecting or capping moiety, usually covalently linked to the N-terminal side of the oligopeptide, which adds to the stability of the prodrug and/or adds to the prevention of internalization of the prodrug into a cell such as a target cell. Such protecting or capping moieties include non-natural amino acids, β-alanyl or -succinyl groups (e.g. WO 96/05863, U.S. Pat. No. 5,962,216). Further stabilizing protecting or capping moieties include diglycolic acid, maleic acid, pyroglutamic acid, glutaric acid, (e.g., WO 00/33888), a carboxylic acid, adipic acid, phthalic acid, fumaric acid, naphthalene dicarboxylic acid, 1,8-naphtyldicarboxylic acid, aconitic acid, carboxycinnamic acid, triazole dicarboxylic acid, butane disulfonic acid, polyethylene glycol (PEG) or an analog thereof (e.g., WO 01/95945), acetic acid, 1- or 2-naphthylcarboxylic acid, gluconic acid, 4-carboxyphenyl boronic acid, polyethylene glycolic acid, nipecotic acid, and isonipecotic acid (e.g., WO 02/00263, WO 02/100353), succinylated polyethylene glycol (e.g., WO 01/91798). A new type of protecting or capping moiety was introduced in WO 2008/120098, being a 1,2,3,4 cyclobutanetetracarboxylic acid. The protecting or capping moiety in WO 02/07770 may be polyglutamic acid, carboxylated dextranes, carboxylated polyethylene glycol or a polymer based on hydroxyprolyl-methacrylamide or N-(2-hydroxyprolyl)methacryloylamide.